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A dual-luciferase reporter system for characterization of small RNA target genes in both mammalian and insect cells

Insect Science 2022年第3期29卷 631-644页

作者： Zhongyuan Deng Yuting Zhang Leyao Li Xingcheng Xie Jinyong Huang Min Zhang Xinzhi Ni Xianchun Li School of Agricultural Sciences Zhengzhou UniversityZhengzhouChina Schooi of Life Sciences Zhengzhou UniversityZhengzhouChina State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina Department of Entomology and BIO5 Institute University of ArizonaTucsonArizonaUSA USDA-ARS Crop Genetics and Breeding Research UnitUniversity of Georgia-Tifton CampusTiftonGeorgiaUSA

MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mrnas)and affect the stability or translational efficiency of the bound *** or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian *** reporter systems,however,are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase(usually Renilla)used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect *** this study,we replaced the SV40 promoter in the psiCHECK-2 reporter vector,which is widely used with mammalian cell lines,with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors,designated psiCHECK-2-TK and psiCHECK-2-AC5.1,*** psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target(7?enz7/a)/reference(firefly)luciferase activity ratios in mammalian(HeLa and HEK293)and insect(Sf9,S2,Helicoverpa zea fat body and ovary)cell lines,while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell ***,psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target,the 3'-untranslated region of heat shock protein 90,in both mammalian and *** cell lines,but psiCHECK-2 failed to do so in *** cell ***,psiCHECK-2-TK with the target sequence,HzMasc(*** Masculinizer),accurately differentiated between *** cell lines with or without the negative regulation factor(miRNA or piRNA)of *** data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.

关键词： Helicoverpa masc miRNA9a mrna degradation promoter psiCHECK-2-TK

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mrna stability in the nucleus

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2014年第5期15卷 444-454页

作者： Han LIU Min LUO Ji-kai WEN Institute of Cancer Stem Cell Dalian Medical University Chongqing Institute of Tuberculosis Prevention and Treatment Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms College of Life SciencesSouth China Agricultural University

Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mrna stability during RNA processing and translation. RNAs, especially mrnas, are relatively vulnerable molecules in living cells for ribonucleases （RNases）. The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mrna stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase Ⅱ in eukaryotes.

关键词： mrna stability Nuclear mrna retention Quality control mrna degradation

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